Name: Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes (Video) | JoVE Journal
Uploaded: 2017-01-10T14:00:00.000+00:00
Duration: 9 min 28 s
Description: Learn to engineer near-infrared fluorescent single-walled carbon nanotubes with biomimetic polymers for molecular sensing. This method enables dopamine detection in living brain tissue using synthetic sensors.

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This work was supported by Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Simons Foundation grant, and a Brain and Behavior Research foundation young investigator grant.

Caution: Please consult all relevant material safety data sheets (SDS) before use. Nanomaterials may have additional hazards compared to their bulk material counterpart. Use all appropriate safety practices including engineering controls (fume hood, noise enclosure) and personal protective equipment (safety glasses, goggles, lab coat, full length pants, closed-toe shoes).
1. Preparation of Buffer, Surfactant, and Polymer Solutions
<ol>
	<li>Preparation of 100 mM NaCl solution
	<ol>
		<li>Dissolve 584 mg of NaCl in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of 3% sodium dodecyl sulfate (SDS) solution
	<ol>
		<li>Dissolve 3 g of SDS in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of 2% sodium cholate (SC) solution
	<ol>
		<li>Dissolve 2 g of sodium cholate hydrate in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of imaging buffer (1x Tris: 20 mM Tris, 100 mM NaCl)
	<ol>
		<li>Dissolve 22.23 g of Tris base and 58.44 g of NaCl in 500 mL of deionized water using a magnetic stir bar and plate.</li>
		<li>Carefully add concentrated HCl until a pH of 8.1 is reached.</li>
		<li>Add deionized water to reach a final volume of 1 L.</li>
	</ol>
	</li>
	<li>Synthesis of RITC-PEG-RITC polymer
	<ol>
		<li>Dissolve amine difunctionalized polyethylene glycol (PEG) (5 kDa or 20 kDa, 0.1 mol/L) and rhodamine isothiocyanate (RITC, 0.2 mol/L) in 1 mL of a 1:1 mixture of dichloromethane and dimethylformamide (DMF).</li>
		<li>Add 0.2 mol/L of N,N-diisopropylethylamine (DIEA).</li>
		<li>After 3 h, precipitate with 10x volume of diethyl ether followed by vacuum filtration.</li>
		<li>Redissolve in DMF and repeat ether precipitation followed by vacuum filtration. 
		NOTE: Other isothiocyanate modified molecules (e.g., fluorescein isothiocyanate, FITC) can be attached to PEG or other amine modified polymers using a similar method.</li>
	</ol>
	</li>
	<li>Preparation of pegylated-DNA (PEG-DNA)
	<ol>
		<li>Combine 100 &#181;L of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (0.5 M, pH 7.0) with 44.9 &#181;L of 5&#39;-thiol-modified DNA (1 mg/10 &#181;L in 0.1 M NaCl) and add to 4.855 mL of deionized water.</li>
		<li>Stir for 1 h.</li>
		<li>Dissolve 500 mg of methoxypolyethylene glycol maleimide in 5 mL of phosphate buffered saline.</li>
		<li>Combine DNA and PEG solutions (10 mL total) and stir for 24 h.</li>
	</ol>
	</li>
</ol>
2. Preparation of Single Walled Carbon Nanotube (SWNTs) Suspensions
<ol>
	<li>Wash of SWNTs to remove catalyst and impurities.
	<ol>
		<li>Add 200-300 mg of unwashed SWNTs into a plastic centrifuge tube containing 45 mL of deionized water.</li>
		<li>Vortex the solution for 2 min and sonicate using a bath sonicator for 5 min. Note that sonicator settings vary from instrument to instrument, so check that the optical density of the SWNT solution increases (i.e., turns black) to ensure that the SWNTs are being dispersed.</li>
		<li>Centrifuge the solution for 20 min at 16,100 x g and discard supernatant.</li>
		<li>Add approximately 45 mL of fresh deionized water.</li>
		<li>Repeat steps 2.1.2-2.1.4 up to 8 times.</li>
		<li>Remove as much water as possible being careful not to disturb pelleted SWNTs and allow SWNT pellet to air dry.</li>
	</ol>
	</li>
	<li>Nucleic acid suspensions of SWNTs
	<ol>
		<li>Dissolve nucleic acids (NA) in 0.1 M NaCl to a concentration of 100 mg/mL.</li>
		<li>Remove static electricity from disposable spatula, microcentrifuge tubes and SWNT stock using an anti-static gun. In a fume hood, add 20 &#181;L of the NA solution to 980 &#181;L of 0.1 M NaCl followed by the addition of 1 mg SWNTs.</li>
		<li>Using an ultrasonicator with 3 mm diameter tip, sonicate the solution for 10 min at 40% amplitude in an ice bath.</li>
		<li>Centrifuge the DNA-SWNT solution TWICE for 90 min at 16,100 x g. If using PEG-DNA, purify out excess and unreacted DNA and PEG using a 100 kDa spin-filter. Add enough PBS to fill the spin-filter and spin at 9,300 x g for 1.5 min, Repeat this wash step 3 times.</li>
		<li>Collect and keep the supernatant, being careful not to disturb the pellet containing CNT bundles and aggregates. Discard the pellet in accordance with institutional hazardous waste procedures appropriate for nanomaterials.</li>
		<li>Measure the solution absorbance at 632 nm using a UV/Vis spectrophotometer to determine approximate concentration of suspended SWNTs using &#949;=0.036 L/cm&#183;mg in accordance with Lambert-Beer&#39;s law and the appropriate dilution factor.</li>
	</ol>
	</li>
	<li>Amphiphilic polymer suspensions of SWNTs
	<ol>
		<li>Remove static electricity from disposable spatula, micro centrifuge tubes, and SWNT stock. In a fume hood, add 5 mg SWNTs to 5 mL of 2% SC solution (alternatively, SDS solution can be used).</li>
		<li>Using an ultrasonicator with 6 mm diameter tip, sonicate the solution for 1 h at 40% amplitude in an ice bath.</li>
		<li>Centrifuge sample at 150,000 x g for 4 h using an ultracentrifuge and carefully collect supernatant.</li>
		<li>Dissolve 1 wt % of amphiphilic polymer (e.g., RITC-PEG-RITC) in SC-SWNT solution.</li>
		<li>Dialyze the polymer-SC-SWNT solution using a 3.5 kDa dialysis membrane against 1 L of deionized water or buffer for 5 days. Change out the water or buffer after hour 2 and hour 4. A larger dialysis membrane can be used, so long as it allows removal of the surfactant of choice, but retention of both the SWNT and amphiphilic polymer.</li>
		<li>Measure the solution absorbance at 632 nm using a UV/Vis spectrophotometer to determine approximate concentration of suspended SWNTs using &#949;=0.036 L/cm*mg in accordance with Lambert-Beer&#39;s law and the appropriate dilution factor.</li>
	</ol>
	</li>
</ol>
3. Preparation of Surface Immobilized SWNT Sensors
<ol>
	<li>Preparation of BSA-biotin and NeutrAvidin stock solutions
	<ol>
		<li>Dissolve 10 mg lyophilized BSA-biotin in 1 mL deionized water to make a 10 mg/mL stock solution and store at 4 &#176;C.</li>
		<li>Dissolve 10 mg NeutrAvidin protein (NAV, deglycosylated avidin protein) in 2 mL of deionized water to make a 5 mg/mL stock solution. Store aliquots at -20 &#176;C. Thawed aliquots can be kept for several days at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare BSA-Biotin coated microscope slides
	<ol>
		<li>Clean a microscope slide and 0.17 mm cover glass (or as appropriate for the microscope objective) with deionized water, followed by methanol, acetone and a final rinse of deionized water.</li>
		<li>Create channels by placing several pieces of double-sided tape approximately 5 mm apart on the clean microscope slide. Seal the channels by taking a long glass coverslip and pressing it onto the top of the double sided tape. Be sure it is centered on the microscope slide so the edges of the cover slip and slide are not flush.</li>
		<li>Add 100 &#181;L of BSA-Biotin stock solution to 900 &#181;L of 1x Tris buffer to a final concentration of 1 mg/mL.</li>
		<li>Flow 50 &#181;L of the BSA-biotin solution into the channel by pipetting the solution into one end and wicking away solution at the other using a tissue. Incubate for 5 minutes followed by 3-5 flushes with 50 &#181;L of 0.1 M NaCl.</li>
		<li>Dilute 40 &#181;L of 5 mg/mL NAV stock in 960 &#181;L 1x Tris buffer to a final concentration of 0.2 mg/mL.</li>
		<li>Flow 50 &#181;L of the NAV solution into the channel by pipetting the solution into one end and wicking away solution at the other using a tissue. Incubate for 2-5 minutes followed by 3-5 flushes with 50 &#181;L of 0.1 M NaCl.</li>
		<li>Dilute stock solutions of suspended SWNTs in imaging buffer to a concentration of 1-10 mg/L and flow 50 &#181;L of the solution into the channel and incubate for 5 min.</li>
		<li>Gently rinse away excess SWNTs using 50 &#181;L of imaging buffer.</li>
	</ol>
	</li>
	<li>Preparation of APTES silanized microscope slides 
	NOTE: APTES silanized slides allow a way to immobilize negatively charged DNA-wrapped SWNTs to the surface of a glass substrate.
	<ol>
		<li>Prepare a 10% solution of (3-aminopropyl)triethoxysilane (APTES) in ethanol.</li>
		<li>Using channels made using the steps outlined in 3.2.2, flow in 100 &#181;L of 1x Tris buffer.</li>
		<li>Flush the channel with APTES solution and incubate for 5 min. Wash with 1x Tris buffer.</li>
		<li>Dilute stock solutions of suspended DNA-SWNTs in imaging buffer to a concentration of 1-10 mg/Land flow 50 &#181;L of the solution into the channel and incubate for 5 min.</li>
		<li>Gently rinse away excess SWNTs using 50 &#181;L of imaging buffer.</li>
	</ol>
	</li>
</ol>
4. Fluorescence Spectroscopy and Microscopy of SWNT Sensors
<ol>
	<li>nIR Fluorescence Microscopy
	<ol>
		<li>Perform imaging of surface immobilized SWNTs using laser excitation and an inverted microscope outfitted with an InGaAs sensor array for imaging.</li>
		<li>Direct the laser beam to enter the back illumination port of an inverted microscope using mirrors on adjustable kinematic mounts and a pair of post-mounted irises set to the port height. Ensure the beam is level and straight by confirming it passes through both irises when placed between subsequent mirrors and before it enters the illumination port. If necessary, adjust the beam height using a periscope assembly. Remove any short-pass heat filters on the illumination port that would attenuate the beam.</li>
		<li>Insert an appropriate filter cube into the microscope to reflect the excitation light into the objective and collect emission light. This typically consists of a dichroic long pass filter with a cutoff above the excitation wavelength, (e.g., 750 LP) and an emission long pass filter (e.g., 850 LP) to further minimize scattered excitation light from hitting the sensor. Additionally, the emission filter can be chosen to be selective for the emission of SWNTs of a particular chirality.</li>
		<li>Perform fine adjustments to the beam alignment by inserting an appropriate alignment cube replacing the objective with two offset, frosted discs containing 1 mm pinholes. Align the beam to the center of both the lower and upper alignment discs.</li>
		<li>Attach a 2D InGaAs sensor array to the side imaging port of the microscope using an appropriate adapter and a 0.5X lens if necessary to accommodate the sensor size.</li>
		<li>Using a 100X oil immersion objective (1.4 NA), apply fluorescence-free immersion oil and place the immobilized SWNT sample onto the microscope stage.</li>
		<li>Raise the objective until the oil contacts the bottom of the cover glass (#1.5, 170 &#181;m thickness). Make adjustments to the objective collar if necessary for imaging conditions, e.g., temperature, glass thickness, etc.</li>
		<li>Slowly raise the objective with the excitation source on and monitor the fluorescence signal of the InGaAs camera. Fluorescence intensity should gradually increase as the focal plane approaches the surface and the immobilized sensors come into focus.</li>
	</ol>
	</li>
	<li>nIR Fluorescence Spectroscopy 
	NOTE: Fluorescence spectroscopy can be performed using the same microscope setup but by directing the collected light out of the microscope body and into a spectrometer and InGaAs linear array detector.
	<ol>
		<li>Using kinematic mounts and mirrors rated for nIR, direct the light path towards the entrance slit of the spectrometer. Focus the light down to a point onto the entrance slit using a focusing lens (e.g., plano-convex, f = 150 mm). This alignment is accomplished by attenuating the laser power to &#60;1 mW and replacing the microscope objective with a 1&#34; mirror and using a 50/50 beam-splitting filter cube. The excitation light will then leave the microscope body through the exit port and can be used to adjust the mirrors and lens to focus the beam onto the entrance slit.</li>
		<li>After replacing the microscope objective and long pass filter, place a well plate onto the stage and record the spectra of an in-focus sample using the spectrometer and InGaAs array.</li>
	</ol>
	</li>
	<li>Measuring the reversible response of (GT)15 DNA-SWNTs to dopamine
	<ol>
		<li>Mount sensor coated channels onto the microscope stage and bring into focus using a 100X oil objective and InGaAs camera. Add 50 &#181;L of 100 &#181;M dopamine solution in phosphate buffered saline (PBS) to the flow channel and record the change in fluorescence intensity.</li>
		<li>Wash out the dopamine solution using phosphate buffered saline and observe the change in fluorescence of the individual SWNT sensors.</li>
	</ol>
	</li>
	<li>Well-plate screening for analyte response of SWNTs sensors 
	NOTE: Screening of different analytes can be performed using a transparent well plate controlled using a motorized stage. An ideal well plate is transparent to visible and IR and has black sidewalls to minimize crosstalk between the wells.
	<ol>
		<li>Pipette equal volumes of suspended SWNT sensor (e.g., 5 mg/L concentration) into each well, enough to cover the bottom of the well uniformly, typically &#62;100 &#181;L for a 96-well plate and &#62;30 &#181;L for a 384-well plate.</li>
		<li>Pipette analytes (e.g., 2 &#181;L, 100 &#181;M final volume) from screening library into the well plates. Prepare each analyte in triplicate to account for potential well-to-well variation, fluctuations of excitation intensity, or temperature.</li>
		<li>Raise the objective (e.g., 20X achroplan, 0.45 NA) while monitoring the emission spectra using the spectrometer and InGaAs array. Optimal position of the objective will put the focal plane approximately in the middle of the sample volume in the well, which should correspond to a maximum in measured intensity.</li>
		<li>For each sample well, record a 1-10 s exposure to collect a spectrum.</li>
		<li>Compare the emission spectra for each well to a control containing just SWNTs without additional analyte to quantify the fluorescence response.</li>
	</ol>
	</li>
</ol>

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<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+recognition+using+corona+phase+complexes+made+of+synthetic+polymers+adsorbed+on+carbon+nanotubes%5BTitle%5D)+AND+%22Nat+nanotechnol%22%5BJournal%5D)">Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.</a> Nat nanotechnol. 8 (12), 959-968 (2013).</li>
<li>Kruss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotransmitter+detection+using+corona+phase+molecular+recognition+on+fluorescent+single-walled+carbon+nanotube+sensors%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Neurotransmitter detection using corona phase molecular recognition on fluorescent single-walled carbon nanotube sensors.</a> J Am Chem Soc. 136 (2), 713-724 (2014).</li>
<li>Salem, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chirality+dependent+corona+phase+molecular+recognition+of+DNA-wrapped+carbon+nanotubes%5BTitle%5D)+AND+%22Carbon%22%5BJournal%5D)">Chirality dependent corona phase molecular recognition of DNA-wrapped carbon nanotubes.</a> Carbon. 97, 147-153 (2016).</li>
<li>Giraldo, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Ratiometric+Sensor+Using+Single+Chirality+Near-Infrared+Fluorescent+Carbon+Nanotubes%3A+Application+to+In+Vivo+Monitoring%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">A Ratiometric Sensor Using Single Chirality Near-Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring.</a> Small. 11 (32), 3973-3984 (2015).</li>
<li>Karousis, N., Tagmatarchis, N., Tasis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+Progress+on+the+Chemical+Modification+of+Carbon+Nanotubes%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">Current Progress on the Chemical Modification of Carbon Nanotubes.</a> Chem Rev. 110 (9), 5366-5397 (2010).</li>
<li>Movia, D., Del Canto, E., Giordani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purified+and+Oxidized+Single-Walled+Carbon+Nanotubes+as+Robust+Near-IR+Fluorescent+Probes+for+Molecular+Imaging%5BTitle%5D)+AND+%22J+Phys+Chem+C%22%5BJournal%5D)">Purified and Oxidized Single-Walled Carbon Nanotubes as Robust Near-IR Fluorescent Probes for Molecular Imaging.</a> J Phys Chem C. 114 (43), 18407-18413 (2010).</li>
<li>Cognet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stepwise+quenching+of+exciton+fluorescence+in+carbon+nanotubes+by+single-molecule+reactions%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Stepwise quenching of exciton fluorescence in carbon nanotubes by single-molecule reactions.</a> Science. 316 (5830), 1465-1468 (2007).</li>
</ol>

The authors have nothing to disclose.

SWNTs are readily suspended in aqueous solution via direct sonication with SDS or ssDNA, as indicated by an increase in optical density provided by the colloidal dispersion of the resulting SWNT-polymer hybrid. SDS and ssDNA disperses and solubilizes bundles of SWNTs by adsorbing onto the SWNT surface through hydrophobic or pi-pi interactions. Additionally, other polymers, such as genomic DNA, amphiphilic polymers, conjugated polymers and lipids, can be adsorbed onto the surface of SWNTs by dialysis of samples suspended ...

Single-walled carbon nanotubes (SWNTs) are atomically thin layers of carbon atoms rolled into long, thin cylinders that exhibit unique electronic and optical properties.1 Such properties include a band-gap producing near infrared (nIR) fluorescence emission via exciton recombination that is highly sensitive to its local environment. The nIR emission of SWNTs falls within the near infrared window in which the penetration depth of light is maximal for biological tissue.2,3 Additionally, SWNTs exhibit several unique features atypical in contrast to organic fluorophores: SWNT exhibit a large Stokes shift, do not photobleach, and do not blink.4 Recently, exploiting these characteristics has led to the development of an assortment of novel molecular sensors with applications to biology.5,6 Unmodified, however, SWNTs are insoluble in water, and obtaining suspensions of individual SWNTs can be a challenge.7,8 Bundling and aggregation of SWNTs in solution can obfuscate their band-gap fluorescence,2 rendering them unsuitable for sensing applications.
Dispersing individual carbon nanotubes in aqueous solution requires modifying their surface to prevent hydrophobicity-driven aggregation.9 While covalent modification can render SWNTs water-soluble,10 as well as impart specific binding chemistry, defect sites in the SWNT lattice reduce or abate their fluorescence emission. Instead, SWNT functionalization can be accomplished by using surfactants, lipids, polymers and DNA9,11-13 that adsorb to the nanotube surface through hydrophobic and pi-pi stacking interactions. The resulting chemical environment surrounding surface-functionalized SWNTs is referred to as its corona phase. Perturbations to the corona phase can have a large impact on excitons traveling on the nanotube surface, causing modulations to SWNT fluorescence emission. It is this sensitive relationship between the corona phase and SWNT fluorescence that can be exploited to develop new molecular sensors by incorporating specific binding modalities onto the large surface area of SWNT. Perturbations to the SWNT corona phase upon binding analyte can lead to changes in the local dielectric environment, charge transfer, or introduce lattice defects, all of which can modulate the fluorescence emission of the SWNTs to serve as a signal transduction mechanism.14 This approach is used in the development of novel fluorescent sensors for the detection of many different classes of molecules including DNA,15,16 glucose17 and small molecules such as ATP,18 reactive oxygen species19 and nitric oxide.20,21 However, these approaches are limited in that they rely on the existence of a known binding modality for the target analyte.
Recently, a more generic approach to designing fluorescent sensors was developed using SWNTs non-covalently functionalized with amphiphilic heteropolymers, phospholipids, and polynucleic acids. These molecules adsorb to carbon nanotube surfaces to produce highly stable suspensions of individual SWNTs22-25 with unique corona phases that can specifically bind proteins26,27 or small molecules including the neurotransmitter dopamine.28-30 Engineering the corona phase to disperse SWNTs and specifically bind target analytes is referred to as corona phase molecular recognition (CoPhMoRe).28 The small size, low toxicity, high stability and unbleaching nIR fluorescence of CoPhMoRe SWNT sensors make them excellent candidates for in vivo sensing for extended time-resolved measurements.6 Recent work has shown their applications in plant tissues for optical detection of reactive nitrogen and oxygen species.31 A particularly exciting application for CoPhMoRe SWNT sensors is the potential for label free detection of neurotransmitters such as dopamine in vivo, where other techniques, such as electrochemical sensing or immunohistochemistry, suffer from a lack of spatial resolution, temporal resolution, and specificity.
Designing and discovering CoPhMoRe SWNT sensors has so far been restrained by the size and chemical diversity of the dispersant library, limiting the likelihood of finding a sensor for a particular target. To date, researchers have only scratched the surface of available conjugated, co-block, biological and biomimetic polymers that could serve as functionally active dispersants for SWNT sensors. Here, we present different methods for both dispersing SWNTs and characterizing their fluorescence for high throughput screening and for single SWNT sensor analysis. Specifically, we outline the procedure for coating SWNTs with polynucleic acid oligomers using direct sonication as well as how to functionalize SWNT with amphiphilic polymers through surfactant exchange by dialysis. We use (GT)15-DNA and polyethylene glycol functionalized with rhodamine isothiocyanate (RITC-PEG-RITC) as examples. We demonstrate the use of (GT)15-DNA SWNTs as a CoPhMoRe sensor for the detection of dopamine. Lastly, we outline procedures for performing single molecule sensor measurements, which can be used for characterization or single molecule sensing.

<table><tbody><tr><td>sodium chloride</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L6026</td><td></td></tr><tr><td>sodium cholate hydrate</td><td>Sigma Aldrich</td><td>C6445</td><td></td></tr><tr><td>tris base (Trizma base)</td><td>Sigma Aldrich</td><td>93362</td><td></td></tr><tr><td>hydrochloric acid</td><td>Fisher Scientific</td><td>A144-212</td><td></td></tr><tr><td>Amine-PEG-amine,NH&lt;sub&gt;2&lt;/sub&gt;-PEG-NH&lt;sub&gt;2&lt;/sub&gt;</td><td>Nanocs Inc</td><td>PG2-AM-5k</td><td></td></tr><tr><td>rhodamine B isothiocyanate</td><td>Sigma Aldrich</td><td>283924</td><td></td></tr><tr><td>fluorescein isothiocyanate</td><td>Sigma Aldrich</td><td>F7250</td><td></td></tr><tr><td>dichloromethane</td><td>Sigma Aldrich</td><td>676853</td><td></td></tr><tr><td>dimethylformamide</td><td>Sigma Aldrich</td><td>D4551</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td></td></tr><tr><td>diethyl ether</td><td>Sigma Aldrich</td><td>673811</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma Aldrich</td><td>C4706&amp;nbsp;</td><td></td></tr><tr><td>5&amp;rsquo;-thiol-modified DNA&amp;nbsp;</td><td>Integrated DNA Technologies</td><td></td><td></td></tr><tr><td>methoxypolyethylene glycol maleimide</td><td>Sigma Aldrich</td><td>63187</td><td></td></tr><tr><td>100 kDa spin filters</td><td>Millipore</td><td></td><td></td></tr><tr><td>HiPCO Super purified single walled carbon nanotubes</td><td>Integris</td><td>HiPco SuperPurified</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Sigma Aldrich</td><td>P5493</td><td></td></tr><tr><td>anti static gun</td><td>Milty</td><td>Milty Zerostat 3</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5415 D</td><td></td></tr><tr><td>ultra sonicator</td><td>Cole Parmer</td><td>CV18</td><td></td></tr><tr><td>dialysis cassettes</td><td>Thermo scientific</td><td>Slide-A-Lyzer G2 87722</td><td></td></tr><tr><td>BSA-biotin</td><td>Thermo scientific</td><td>29130</td><td></td></tr><tr><td>Neutravidin protein</td><td>Thermo scientific</td><td>31000</td><td></td></tr><tr><td>(3-Aminopropyl)triethoxysilane (APTES)</td><td>Sigma Aldrich</td><td>440140</td><td></td></tr><tr><td>inverted microscope</td><td>Zeiss</td><td>Axio Observer.Z1</td><td></td></tr><tr><td>kinematic mirrors</td><td>ThorLabs</td><td>KM200-E03</td><td></td></tr><tr><td>periscope</td><td>ThorLabs</td><td>RS99</td><td></td></tr><tr><td>immersion oil</td><td>Zeiss</td><td>Immersol 518f</td><td></td></tr><tr><td>100X objective</td><td>Zeiss</td><td>Plan-apochromat 100X oil, 1.4NA, PH3, 420791-9911-000</td><td></td></tr><tr><td>20X objective</td><td>Zeiss</td><td>N-Achroplan 0.45 NA, 420953-9901-000&amp;nbsp;</td><td></td></tr><tr><td>cover glass</td><td>Healthrow Scientific</td><td>HS159879H</td><td></td></tr><tr><td>dopamine hydrochloride</td><td>Sigma Aldrich</td><td>H8502&amp;nbsp;</td><td></td></tr><tr><td>infrared 2D array camera</td><td>Princeton Instruments</td><td>NIRvana</td><td></td></tr><tr><td>infrared 1D sensor array</td><td>Princeton Instruments</td><td>PyLoN IR</td><td></td></tr><tr><td>nIR spectrograph</td><td>Princeton Instruments</td><td>SCT-320</td><td></td></tr><tr><td>planoconvex lens</td><td>ThorLabs</td><td>LA1384</td><td></td></tr><tr><td>well plates (glass bottom)</td><td>Corning</td><td>4580</td><td></td></tr></tbody></table>

engineering molecular recognition with bio-mimetic polymers on single walled carbon nanotubes

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

SWNTs were suspended in aqueous solution using both surfactants and amphiphilic polymers by direct sonication and by dialysis exchange. Figure 1 shows SWNTs, grown using the iron carbonyl catalyzed method (HiPCO), suspended using SC, RITC-PEF20-RITC, and (GT)15-DNA. The optical density of a SWNTs with SDS (or polymer) increases dramatically after sonication and decreases upon removal of aggregates and contaminants through purification by centrifugation (<strong...

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

The overall goal of this procedure is to prepare near-infrared fluorescent single walled carbon nanotubes for molecular sensing that have been functionalized using biomimetic polymers. These nanosensors can enable time resolved imaging in living tissue. For example, they can be used to detect dopamine in the brain.The main advantage of these sensors is their inherent near infrared fluorescence, which enables imaging deeper into tissue. Synthetic sensors enable the detection of molecules without natural molecular recognition elements making this generic method of sensors development especially useful for small molecule detection. To prepare a nucleic acid suspension of SWNTs, first dissolve nucleic acids in 0.1 molar sodium chloride to a concentration of 100 milligrams per milliliter.In a fume hood, remove static electricity from the disposable spatula, microcentrifuge tubes, and the SWMT stock, using an antistatic gun. Next, add 20 microliters of the nucleic acid solution to 980 microliters of 0.1 molar sodium chloride. Then add 1 milligram of washed SWNTs to the resulting nucleic acid solution before sonicating the mixture.Positioning the probe tip during sonication is critical. I

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Bioengineering

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Polymer Microarrays for High Throughput Discovery of Biomaterials

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Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

A Polyaniline-based Sensor of Nucleic Acids

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

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Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

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Biofunctionalization of Magnetic Nanomaterials

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

Learn to engineer near-infrared fluorescent single-walled carbon nanotubes with biomimetic polymers for molecular sensing. This method enables dopamine detection in living brain tissue using synthetic sensors.

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

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